Cytoplasmic antiproteinase-2 and cytoplasmic antiproteinase-3 and coding sequences

ABSTRACT

Cytoplasmic Antiproteinase-2 and Cytoplasmic Antiproteinase-3 nucleic acids and serine protease inhibitor proteins encoded thereby are useful in the purification of proteins and in the treatment of inflammatory diseases and diseases involving apoptosis.

This application is a divisional of Ser. No. 08/385,500, filed Feb. 8,1995, (pending) which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Serine proteases play a critical role in many physiological processes.For example, serine proteases are involved in blood coagulation,fibrinolysis, complement activation, and inflammation. The catalyticactivity of these serine proteases is often regulated by members of asuper-family of serine protease inhibitors called serpins. Serpins actto regulate the activity of serine proteases by bindingstoichiometrically to the active sites of serine proteases and thusinactivating the enzymes. See Cartell, et al. (1987) Cold Spring HarborSymposia, vol. LII, pp. 527-535 for a description of the generalstructural characteristics of serpins.

Many serpins are extracellular proteins which regulate extracellularprocesses such as blood coagulation, fibrinolysis and complementactivation. In addition, there is a family of serpins structurallyrelated to ovalbumin which lack secretory peptide sequences and whichmay function, in part, intracellularly. Several of this latter group ofserpins are believed to play an important role in regulating serineprotease activity in inflammation. Elastase inhibitor is an example ofsuch a serpin which functions to regulate the activity of neutrophilelastase. Neutrophil elastase is stored in the azurophil granules ofneutrophils, monocytes and macrophages, and degrades both phagocytizedand extracellular substrates. Regulation of neutrophil elastase isimportant both in host defense mechanisms and also in the pathology ofdiseases such as arthritic joint diseases.

Interleukin-1β converting enzyme (ICE) is an another example of acysteine protease that plays an important role in inflammation. ICE isresponsible for the activation of interleukin-1β, which is a criticalcytokine in the inflammatory process. Serpins which inhibit ICE maytherefore play an important role in inflammation. One such serpin is aviral protein encoded by the cowpox virus crmA gene. It is believed thatexpression of crmA protein inhibits ICE and thereby blocks migration ofinflammatory cells in cowpox lesions. (See Ray, C. A., et. al. (1992)Cell 69:597-604.) Isolated cellular serpins that inhibit ICE in asimilar manner to the crmA protein can be useful in the modulation ofthe inflammatory response. Agents that modulate the inflammatoryresponse can be used in the treatment of a variety of inflammatorydiseases, such as rheumatoid arthritis.

ICE is but one member of a family of serine proteases that playimportant roles in normal physiology and in pathophysiology. Forexample, another member of the ICE family, Ich-1, is involved inregulation of apoptosis. Furthermore, evidence is accumulating thatregulation of apoptosis plays a role in a variety of different diseases,including cancer. Therefore, isolated serpin molecules which inhibitIch-1 could be used to regulate apoptosis and treat a number ofdiseases.

Isolated serpin molecules are also useful in the purification of avariety of proteins for use in medicine and industry. Proteindegradation during purification by endogenous serine proteases is acommon problem. Isolation of serpins inhibiting different serineproteases is useful to improve the purification of many differentproteins. These and other needs are addressed by the present invention.

SUMMARY OF THE INVENTION

The present invention provides isolated nucleic acid molecules encodingmammalian CAP-2 and CAP-3 protein. The CAP-2 and CAP-3 proteins of theinvention are homologous to the amino acid sequence depicted in SEQ IDNO:2 and SEQ ID NO:4, respectively, including the amino acid sequencedepicted in SEQ ID NO:2 or SEQ ID NO:4, or an allelic variant thereof.Typically the CAP-2 or CAP-3 protein and polypeptides thereof will becapable of inhibiting serine protease activity. The isolated nucleicacid molecule, e.g., DNA or RNA, may encode human CAP-2 or CAP-3protein.

Thus, in another aspect the invention includes isolated mammalian CAP-2protein which is homologous to the amino acid sequence depicted in SEQID NO:2 and inhibits serine protease activity, as well as isolatedmammalian CAP-3 protein homologous to the amino acid sequence of SEQ IDNO:4. In exemplary embodiments described herein the CAP-2 and CAP-3proteins are human, e.g., the protein of SEQ ID NO: 2 or SEQ ID NO:4, oran allelic variant thereof.

In other embodiments the invention provides expression vectors having asoperably linked elements a transcriptional promoter, a DNA segmentencoding a mammalian CAP-2 or CAP-3 protein wherein said protein ishomologous to the amino acid sequence depicted in SEQ ID NO:2 or SEQ IDNO:4 or an allelic variant, and inhibits serine protease activity, and atranscriptional terminator. Cultured host cells are also provided whichare transformed or transfected with these expression vectors. Preferablythe host cell is a mammalian cell.

In other aspects the invention provides methods for purifying a proteinin a solution which contains a protease. The solution containing theprotein of interest is exposed to a mammalian CAP-2 or a CAP-3 serineprotease inhibitor, for example, that which has been immobilized to anaffinity column, whereby the protease binds to the inhibitor and theactivity of the protease is inhibited. One or more subsequent separationor purification steps can be performed in the presence of said serineprotease inhibitor to obtain said purified protein.

In another embodiment methods are provided for producing a mammalianCAP-2 or CAP-3 polypeptide by growing eukaryotic cells, especiallymammalian cells, transformed or transfected with a DNA construct whichcomprises an operably linked transcriptional promoter, a DNA segmentencoding a mammalian CAP-2 or CAP-3 polypeptide which inhibits serineprotease activity, and a transcriptional terminator. The CAP-2 or CAP-3polypeptide is preferably homologous to the amino acid sequence of SEQID NO:2 or SEQ ID No:4, respectively. The cells are cultured underconditions whereby the CAP-2 or CAP-3 encoding DNA segment is expressed.The mammalian CAP-2 or CAP-3 polypeptide can then be isolated from thehost cells, e.g., by affinity purification or similar procedure.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present invention provides for isolated cytoplasmic antiproteinase-2(CAP-2) proteins, for isolated cytoplasmic antiproteinase-3 (CAP-3)proteins, polypeptides thereof, and for isolated nucleic acids encodingthese proteins and polypeptides. The isolated CAP-2 and CAP-3 nucleicacid and protein compositions can be used in a number of applications.For instance, protease degradation during protein purification is acommon problem in protein chemistry. CAP-2 and CAP-3 serpins can be usedin the purification of a variety of proteins, including those of knownimportance in medicine and industry. As described herein, CAP-2 andCAP-3 nucleic acid and protein compositions are also useful in thetreatment of inflammatory diseases and in the treatment of diseasesinvolving apoptosis. In addition, these compositions can be used in invitro diagnostic procedures for these diseases.

By "isolated" CAP-2 or CAP-3 is meant to refer to CAP-2 or CAP-3 whichis in other than its native environment, and includes, for example,substantially purified CAP-2 and CAP-3 as defined herein. Moregenerally, isolated is meant to include CAP-2 or CAP-3 as a heterologouscomponent of a cell or other system. For example, CAP-2 or CAP-3 may beexpressed by a cell transfected with a DNA construct which encodes CAP-2or CAP-3, and then separated from the cell. Thus, in this context, theenvironment of isolated CAP-2 or CAP-3 is not as it occurs in its nativestate, particularly when it is present in a system as an exogenouscomponent.

The predicted amino acid sequences of human CAP-2 and CAP-3 proteins aredepicted in Seq. ID No. 2 and Seq. ID No. 4, respectively. The predictedamino acid sequences of CAP-2 and CAP-3 are 374 and 376 amino acids,respectively. Both human proteins have a predicted molecular weight ofabout 42 kDa.

Human CAP-2 and CAP-3 proteins share some amino acid sequence identitywith other members of the ovalbumin branch of the serpin superfamily ofproteinase inhibitors. Relatedness between these group of proteins wascalculated using the NBRF ALIGN program. Using this program, the CAP-2and CAP-3 human proteins have 68% and 63% identity, respectively, withthe human cytoplasmic antiproteinase (CAP-1) protein sequence isolatedfrom human placenta. Morgenstern et al., Biochem. 33:3432-3441 (1994).In addition, CAP-2 shows 63% amino acid sequence identity to CAP-3.Human CAP-2 and CAP-3 also exhibit a degree of amino acid sequenceidentity to other human members of the ovalbumin family of cytoplasmicserpins. For instance, CAP-2 has 51% identity and CAP-3 has 49% identitywith elastase inhibitor. CAP-2 and CAP-3 have 46% and 45% identity withplasminogen activator inhibitor-2, respectively. In addition, CAP-2 has46% identity and CAP-3 has 45% identity with squamous cell carcinomaantigen.

CAP-2 and CAP-3 lack a typical N-terminal cleavable signal sequence thatis present in many other members of the serpin superfamily. CAP-2 andCAP-3 also lack a C-terminal extension which is present in many serpins.CAP-2 and CAP-3 both have a serine corresponding to position 375 of α₁-proteinase inhibitor in place of a highly conserved Asn found among theserpins distantly related to the ovalbumin family. CAP-1 and CAP-2 alsohave potential N-glycosylation consensus motifs (N-X-T/S) starting atAsh⁸ and Ash⁷⁸ of CAP-2 and Asn⁶ and Asn²³ of CAP-3.

CAP-2 polypeptides typically show substantial sequence identity orhomology to the amino acid sequence of Seq ID No. 2. Similarly, CAP-3polypeptides typically show substantial sequence identity to the aminoacid sequence of Seq. ID No. 4. As applied to these polypeptides andpeptides thereof, the terms "substantial sequence identity" or"homology" or "homologous" mean that two amino acid sequences, whenoptimally aligned, such as by the programs GAP or BESTFIT using defaultgap penalties, share at least 80 percent sequence identity, preferablyat least 90 percent sequence identity, more preferably at least 95percent sequence identity or more. "Percentage amino acid identity" or"percentage amino acid sequence identity" refers to a comparison of theamino acids of two polypeptides which, when optimally aligned, haveapproximately the designated percentage of the same amino acids.Preferably, residue positions which are not identical differ byconservative amino acid substitutions. For example, the substitution ofamino acids having similar chemical properties such as charge orpolarity are not likely to substantially effect the properties of aprotein. Examples include glutamine for asparagine or glutamic acid foraspartic acid.

The terms "CAP-2 protein" and "CAP-3 protein" refer not only to theamino acid sequences disclosed herein, but also to other proteins thatare allelic or species variants of these amino acid sequences. It isalso understood that these terms include nonnatural mutations introducedby deliberate mutation using recombinant technology such as single sitemutation or by excising short sections of DNA encoding CAP-2 or CAP-3proteins or by substituting new amino acids or adding new amino acids.Such minor alterations substantially maintain the immunoidentity of theoriginal molecule and/or its biological activity. The biologicalproperties of the altered proteins can be determined by expressing theprotein in an appropriate cell line and by determining the ability ofthe protein to inhibit designated serine proteases. The biologicalactivity of CAP-2 can be determined by its ability to inhibit serineproteases, for example, those with trypsin-like specificity. Thebiological activity of CAP-3 can be determined, for example, by itsability to inhibit proteases in the ICE family. Particular proteinmodifications considered minor would include substitution of amino acidsof similar chemical properties, e.g., glutamic acid for aspartic acid orglutamine for asparagine.

By aligning a protein optimally with the protein of Seq. ID No. 2 orSeq. ID No. 4, and by using immunoassays as described herein todetermine immunoidentity, one can readily determine the proteincompositions of the invention. For example, CAP-2 proteins fromdifferent mammalian species, e.g., other primate species, are typicallyspecifically immunoreactive with antibodies raised to the CAP-2 proteindepicted in Seq. ID No. 2, whereas CAP-3 proteins from differentmammalian species, e.g., other primate species, are typicallyspecifically immunoreactive with antibodies raised to the CAP-3 proteindepicted in Seq. ID No. 4.

In other embodiments the present invention provides isolated nucleicacid molecules which encode the CAP-2 or CAP-3 proteins. The term"isolated" as applied to nucleic acid molecules means those which areseparated from their native environment, and preferably free ofnon-CAP-2 or non-CAP-3 DNA or coding sequences with which they arenaturally associated. The term "nucleic acids", as used herein, refersto either DNA or RNA. "Nucleic acid molecule" or "polynucleotidesequence" refers to a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5' to the 3'end. The nucleic acid molecules of the invention, whether RNA, cDNA, orgenomic DNA, may be isolated from natural sources or may be prepared invitro. The nucleic acids may be present in transformed or transfectedwhole cells, in a transformed or transfected cell lysate, or in apartially purified or substantially pure form.

The nucleic acid molecules of the invention are typically identical toor show substantial sequence identity or homology (determined asdescribed herein) to the nucleic acid molecules having sequences of SeqID. Nos. 1 and 3 or the complements thereof. The nucleic acid moleculesinclude those which are equivalent to native or allelic sequences due tothe degeneracy of the genetic code as well as sequences which areintroduced to provide codon preference in a specific host cell. Nucleicacids encoding mammalian CAP-2 and CAP-3 proteins will typicallyhybridize to the nucleic acid sequences of Seq. ID No. 1 or Seq. ID No.3, respectively, under stringent hybridization conditions. Lessstringent hybridization conditions may also be selected. Generally,stringent conditions are selected to be about 5° C. lower than thethermal melting point (Tm) for the specific sequence at a defined ionicstrength and pH. The Tm is the temperature (under defined ionic strengthand pH) at which 50% of the target sequence hybridizes to a perfectlymatched probe. Typically, stringent conditions will be those in whichthe salt concentration is at least about 0.02 molar at pH 7 and thetemperature is at least about 60° C. As other factors may significantlyaffect the stringency of hybridization, including, among others, basecomposition and size of the complementary strands, the presence oforganic solvents and the extent of base mismatching, the combination ofparameters is more important than the absolute measure of any one. Thus,the phrase "selectively hybridizing to" refers to a nucleic acid probethat hybridizes, duplexes or binds preferentially to a particular targetDNA or RNA sequence when the target sequences are present in apreparation of total cellular DNA or RNA. "Complementary" or "target"nucleic acid sequences refer to those nucleic acid sequences whichselectively hybridize to a nucleic acid probe. For discussions ofnucleic acid probe design and annealing conditions, see, for example,Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed.), Vols.1-3, Cold Spring Harbor Laboratory, (1989) or Current Protocols inMolecular Biology, F. Ausubel et al., ed., Greene Publishing andWiley-Interscience, New York (1987), each of which is incorporatedherein by reference.

Techniques for manipulation of nucleic acids encoding CAP-2 and CAP-3proteins such as subcloning nucleic acid sequences encoding polypeptidesinto expression vectors, labelling probes, DNA hybridization, and thelike are described generally in Sambrook, supra.

There are various methods of isolating nucleic acid molecules encodingCAP-2 or CAP-3 proteins. For example, DNA is isolated from a genomic orcDNA library using labeled oligonucleotide probes having sequencescomplementary to the sequences disclosed herein (Seq. ID No. 1 and Seq.ID No. 3). Full-length probes may be used, or oligonucleotide probes mayalso be generated by comparison of the sequences of Seq. ID Nos. 1 and3. Such probes can be used directly in hybridization assays to isolateDNA encoding CAP-2 or CAP-3 proteins. Alternatively, probes can bedesigned for use in amplification techniques such as PCR (Mullis et al.,U.S. Pat. Nos. 4,683,195 and 4,683,202, incorporated herein byreference), and DNA encoding CAP-2 and CAP-3 proteins may be isolated byusing methods such as PCR. Nucleic acid probes may be DNA or RNAfragments. DNA fragments can be prepared, for example, by digestingplasmid DNA, or by use of PCR, or synthesized by either thephosphoramidite method described by Beaucage and Carruthers, TetrahedronLett. 22:1859-1862 (1981), or by the triester method according toMatteucci, et al., J. Am. Chem. Soc., 103:3185 (1981), both incorporatedherein by reference. A double stranded fragment may then be obtained, ifdesired, by annealing the chemically synthesized single strands togetherunder appropriate conditions or by synthesizing the complementary strandusing DNA polymerase with an appropriate primer sequence. Where aspecific sequence for a nucleic acid probe is given, it is understoodthat the complementary strand is also identified and included. Thecomplementary strand will work equally well in situations where thetarget is a double-stranded nucleic acid.

To prepare a cDNA library, mRNA is isolated from tissue such as humanplacenta which expresses CAP-2 or CAP-3 protein. cDNA is prepared fromthe mRNA and ligated into a recombinant vector. The vector istransfected into a recombinant host for propagation, screening andcloning. Methods for making and screening cDNA libraries are well known.See Gubler and Hoffman, Gene 25:263-269 (1983) and Sambrook, et al.,supra.

For a genomic library, the DNA is extracted from tissue and eithermechanically sheared or enzymatically digested to yield fragments ofabout 12-20 kb. The fragments are then separated by gradientcentrifugation from undesired sizes and are constructed in bacteriophagelambda vectors. These vectors and phage are packaged in vitro, asdescribed in Sambrook, et al. Recombinant phage are analyzed by plaquehybridization as described in, e.g., Benton and Davis, Science,196:180-182 (1977). Colony hybridization is carried out as generallydescribed in, e.g., Grunstein et al. Proc. Natl. Acad. Sci. USA.,72:3961-3965 (1975).

DNA encoding a CAP-2 or CAP-3 protein is identified in either cDNA orgenomic libraries by its ability to hybridize with nucleic acid probes,for example on Southern blots, and these DNA regions are isolated bystandard methods familiar to those of skill in the art. See Sambrook, etal.

Various methods of amplifying target sequences, such as the polymerasechain reaction, can also be used to prepare nucleic acids encoding CAP-2or CAP-3 proteins. PCR technology is used to amplify such nucleic acidsequences directly from mRNA, from cDNA, and from genomic libraries orcDNA libraries. The isolated sequences encoding CAP-2 or CAP-3 proteinsmay also be used as templates for PCR amplification.

In PCR techniques, oligonucleotide primers complementary to the two 3'borders of the DNA region to be amplified are synthesized. Thepolymerase chain reaction is then carried out using the two primers. SeePCR Protocols: A Guide to Methods and Applications. Innis, M., Gelfand,D., Sninsky, J. and White, T., eds., Academic Press, San Diego (1990).Primers can be selected to amplify the entire regions encoding afull-length CAP-2 or CAP-3 protein or to amplify smaller DNA segments asdesired.

PCR can be used in a variety of protocols to isolate cDNAs encodingCAP-2 or CAP-3 proteins. In these protocols, appropriate primers andprobes for amplifying DNA encoding CAP-2 or CAP-3 proteins are generatedfrom analysis of the DNA sequences listed herein. Once such regions arePCR-amplified, they can be sequenced and oligonucleotide probes can beprepared from sequence obtained. These probes can then be used toisolate DNA's encoding CAP-2 or CAP-3 proteins, similar to the procedureused in example 2 herein. CAP-2 and CAP-3 proteins can be isolated froma variety of different tissues using this procedure.

Oligonucleotides for use as probes are chemically synthesized accordingto the solid phase phosphoramidite triester method first described byBeaucage and Carruthers, Tetrahedron Lett., 22:1859-1862 (1981), usingan automated synthesizer, e.g., as described in Needham-VanDevanter, etal., Nucleic Acids Res., 12:6159-6168 (1984). Purification ofoligonucleotides is by either native acrylamide gel electrophoresis orby anion-exchange HPLC as described in Pearson and Regnier, J. Chrom.,255:137-149 (1983). The sequence of the synthetic oligonucleotide can beverified using the chemical degradation method of Maxam and Gilbert,Meth. Enzymol., 65:499-560 (1984).

Other methods known to those of skill in the art may also be used toisolate DNA molecules encoding CAP-2 or CAP-3 proteins. See Sambrook, etal. for a description of other techniques for the isolation of DNAencoding specific protein molecules. Thus, the present inventionincludes nucleotide sequences that have substantial sequence identity orhomology to the CAP-2 and CAP-3 nucleotide sequences described in SEQ IDNOs 2 and 4. For substantial sequence identity or homology thepolynucleotide comprises a sequence that has at least 80 percentsequence identity, preferably at least 90 percent sequence identity, andmore preferably at least 95 percent sequence identity. The comparison ismade to a reference sequence over a comparison window of at least 20nucleotide positions, frequently over a window of at least 25-50nucleotides, wherein the percentage of sequence identity is calculatedby comparing the reference sequence to the polynucleotide sequence,which may include deletions or additions which total 20 percent or lessof the reference sequence over the window of comparison. The referencesequence may be a subset of a larger sequence, for example, as a segmentof the human CAP-2 or CAP-3 sequences described herein. Optimalalignment of sequences for aligning a comparison window may be conductedaccording to the local homology algorithm of Smith and Waterman (1981)Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needlemanand Wunsch (1970) J. Mol. Biol. 48:443, by the search for similaritymethod of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. (USA)85:2444, or by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software PackageRelease 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.).

Once DNA encoding CAP-2 or CAP-3 proteins or a homologous sequence isisolated and cloned, CAP-2 or CAP-3 proteins or a homologous protein canbe expressed in a variety of recombinantly engineered cells. Numerousexpression systems are available for expression of DNA encoding CAP-2 orCAP-3 proteins. The expression of natural or synthetic nucleic acidsencoding CAP-2 or CAP-3 proteins will typically be achieved by operablylinking the DNA to a promoter (which is either constitutive orinducible) within an expression vector. By expression vector is meant aDNA molecule, linear or circular, that comprises a segment encoding aCAP-2 or CAP-3 protein or polypeptide of interest, operably linked toadditional segments that provide for its transcription. Such additionalsegments include promoter and terminator sequences. An expression vectormay also include one or more origins of replication, one or moreselectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both. The term "operably linked" indicates thatthe segments are arranged so that they function in concert for theirintended purposes, e.g., transcription initiates in the promoter andproceeds through the coding segment to the terminator. See Sambrook etal., supra.

A variety of procaryotic expression systems may be used to express CAP-2or CAP-3 proteins. Examples include E. coli, Bacillus, Streptomyces, andthe like. For example, CAP-2 and CAP-3 proteins may be expressed in E.coli.

Expression vectors can be constructed which contain a promoter to directtranscription, a ribosome binding site, and a transcriptionalterminator. Examples of regulatory regions suitable for this purpose inE. coli are the promoter and operator region of the E. coli tryptophanbiosynthetic pathway as described by Yanofsky, J. Bacteriol.,158:1018-1024 (1984) and the leftward promoter of phage lambda (Pλ) asdescribed by Herskowitz and Hagen, Ann. Rev. Genet., 14:399-445 (1980).The inclusion of selection markers in DNA vectors transformed in E. coliis also useful. Examples of such markers include genes specifyingresistance to ampicillin, tetracycline, or chloramphenicol. Vectors usedfor expressing foreign genes in bacterial hosts will generally contain aselectable marker, such as a gene for antibiotic resistance, and apromoter which functions in the host cell. Plasmids useful fortransforming bacteria include pBR322 (Bolivar et al., Gene 2:95-113(1977)), the pUC plasmids (Messing, Meth. Enzymol. 101:20-77 (1983),Vieira and Messing, Gene 19:259-268 (1982)), pCQV2 (Queen, ibid.), andderivatives thereof. Plasmids may contain both viral and bacterialelements. Methods for the recovery of the proteins in biologicallyactive form are discussed in U.S. Pat. Nos. 4,966,963 and 4,999,422,which are incorporated herein by reference. See Sambrook, et al. for adescription of other prokaryotic expression systems.

CAP-2 or CAP-3 proteins produced by prokaryotic cells may notnecessarily fold properly. During purification from E. coli, theexpressed protein may first be denatured and then renatured. This can beaccomplished by solubilizing the bacterially produced proteins in achaotropic agent such as guanidine HCl and reducing all the cysteineresidues with a reducing agent such as beta-mercaptoethanol. The proteinis then renatured, either by slow dialysis or by gel filtration. SeeU.S. Pat. No. 4,511,503, incorporated herein by reference. Detection ofthe expressed protein is achieved by methods such as radioimmunoassay,Western blotting techniques or immunoprecipitation.

For expression in eukaryotes, host cells for use in practicing thepresent invention include mammalian, avian, plant, insect, and fungalcells. Fungal cells, including species of yeast (e.g., Saccharomycesspp., Schizosaccharomyces spp.) or filamentous fungi (e.g., Aspergillusspp., Neurospora spp.) may be used as host cells within the presentinvention. Strains of the yeast Saccharomyces cerevisiae can be used. Asexplained briefly below, CAP-2 and CAP-3 proteins can be expressed inthese eukaryotic systems.

Recombinantly produced CAP-2 or CAP-3 proteins can be directed into thesecretory pathway of the host cell in order to facilitate purification,by using at least one signal sequence operably linked to the DNAsequence of interest. Examples of such signals include the alpha factorsignal sequence (pre-pro sequence; Kurjan and Herskowitz, Cell 30:933-943 (1982); Kurjan et al., U.S. Pat. No. 4,546,082; Brake, U.S. Pat.No. 4,870,008), the PHO5 signal sequence (Beck et al., WO 86/00637), theBAR1 secretory signal sequence (MacKay et al., U.S. Pat. No. 4,613,572;MacKay, WO 87/002670), the SUC2 signal sequence (Carlson et al., Mol.Cell. Biol. 3: 439-447 (1983)), the α-1-antitrypsin signal sequence(Kurachi et al., Proc. Natl. Acad. Sci. USA 78: 6826-6830 (1981)), theα-2 plasmin inhibitor signal sequence (Tone et al., J. Biochem. (Tokyo)102: 1033-1042 (1987)) and the tissue plasminogen activator leadersequence (Pennica et al., Nature 301: 214-221 (1983)). Alternatively, asecretory signal sequence may be synthesized according to the rulesestablished, for example, by yon Heinje (Eur. J. Biochem. 133: 17-21(1983); J. Mol. Biol. 184: 99-105, (1985); Nuc. Acids Res. 14: 4683-4690(1986)).

Signal sequences may be used singly or may be combined. For example, afirst signal sequence may be used singly or in combination with asequence encoding the third domain of Barrier (described in U.S. Pat.No. 5,037,743, incorporated by reference herein in its entirety). A DNAsegment encoding the third domain of Barrier may be positioned in properreading frame 3' of the CAP-2 or CAP-3 DNA sequence of interest or 5' tothe DNA sequence and in proper reading frame with both the signalsequence and the CAP-2 or CAP-3 DNA sequence of interest.

Suitable yeast vectors for use in the present invention include YRp7(Struhl et al., Proc. Natl. Acad. Sci. USA 76: 1035-1039 (1978)), YEp13(Broach et al., Gene 8: 121-133 (1979)), POT vectors (Kawasaki et al,U.S. Pat. No. 4,931,373, which is incorporated by reference herein),pJDB249 and pJDB219 (Beggs, Nature 275:104-108 (1978)) and derivativesthereof. Such vectors will generally include a selectable marker, whichmay be one of any number of genes that exhibit a dominant phenotype forwhich a phenotypic assay exists to enable transformants to be selected.Preferred selectable markers are those that complement host cellauxotrophy, provide antibiotic resistance or enable a cell to utilizespecific carbon sources, and include LEU2 (Broach et al., ibid.), URA3(Botstein et al., Gene 8: 17 (1979)), HIS3 (Struhl et al., ibid.) orPOT1 (Kawasaki et al., ibid.). Another suitable selectable marker is theCAT gene, which confers chloramphenicol resistance on yeast cells.

Examples of promoters for use in yeast include promoters from yeastglycolytic genes (Hitzeman et al., J. Biol. Chem. 255: 12073-12080(1980); Alber and Kawasaki, J. Mol. Appl. Genet. 1: 419-434 (1982);Kawasaki, U.S. Pat. No. 4,599,311) or alcohol dehydrogenase genes (Younget al., in Genetic Engineering of Microorganisms for Chemicals,Hollaender et al., (eds.), p. 355, Plenum, New York (1982); Ammerer,Meth. Enzymol. 101: 192-201 (1983)). The TPI1 promoter (Kawasaki, U.S.Pat. No. 4,599,311, 1986) and the ADE2-4^(c) promoter (Russell et al.,Nature 304: 652-654 (1983); and EP 284,044 can also be used. Theexpression units may also include a transcriptional terminator. Anexample of such a transcriptional terminator is the TPI1 terminator(Alber and Kawasaki, ibid.).

In addition to yeast, proteins of the present invention can be expressedin filamentous fungi, for example, strains of the fungi Aspergillus(McKnight et al., U.S. Pat. No. 4,935,349, which is incorporated hereinby reference). Examples of useful promoters include those derived fromAspergillus nidulans glycolytic genes, such as the ADH3 promoter(McKnight et al., EMBO J. 4: 2093-2099 (1985)) and the tpiA promoter. Anexample of a suitable terminator is the ADH3 terminator (McKnight etal., ibid.). The expression units utilizing such components are clonedinto vectors that are capable of insertion into the chromosomal DNA ofAspergillus.

Techniques for transforming fungi are well known in the literature, andhave been described, for instance, by Beggs (ibid.), Hinnen et al.(Proc. Natl. Acad. Sci. USA 75: 1929-1933 (1978)), Yelton et al. (Proc.Natl. Acad. Sci. USA 81: 1740-1747 (1984)), and Russell (Nature 301:167-169 (1983)). The genotype of the host cell will generally contain agenetic defect that is complemented by the selectable marker present onthe expression vector. Choice of a particular host and selectable markeris well within the level of ordinary skill in the art.

In addition to fungal cells, cultured mammalian cells may be used ashost cells within the present invention. Examples of cultured mammaliancells for use in the present invention include the COS-1 (ATCC CRL1650), BHK, and 293 (ATCC CRL 1573; Graham et al., J. Gen. Virol. 6:59-72 (1977)) cell lines. An example of a BHK cell line is the BHK 570cell line (deposited with the American Type Culture Collection underaccession number CRL 10314). In addition, a number of other mammaliancell lines may be used within the present invention, including Rat Hep I(ATCC CRL 1600), Rat Hep II (ATCC CRL 1548), TCMK (ATCC CCL 139), Humanlung (ATCC CCL 75.1), Human hepatoma (ATCC HTB-52), Hep G2 (ATCC HB8065), Mouse liver (ATCC CCL 29.1), NCTC 1469 (ATCC CCL 9.1) and DUKXcells (Urlaub and Chasin, Proc. Natl. Acad. Sci USA 77: 4216-4220(1980)).

Mammalian expression vectors for use in carrying out the presentinvention will include a promoter capable of directing the transcriptionof a cloned gene or cDNA. Both vital promoters or cellular promoters canbe used. Viral promoters include the immediate early cytomegaloviruspromoter (Boshart et al., Cell 41: 521-530 (1985)) and the SV40 promoter(Subramani et al., Mol. Cell. aiol. 1: 854-864 (1981)). Cellularpromoters include the mouse metallothionein-1 promoter (Palmiter et al.,U.S. Pat. No. 4,579,821), a mouse V_(K) promoter (Bergman et al., Proc.Natl. Acad. Sci. USA 81: 7041-7045 (1983); Grant et al., Nuc. Acids Res.15: 5496 (1987)), a mouse V_(H) promoter (Loh et al., Cell 33: 85-93(1983)), and the major late promoter from Adenovirus 2 (Kaufman andSharp, Mol. Cell. Biol. 2: 1304-13199 (1982)). Such expression vectorscan also contain a set of RNA splice sites located downstream from thepromoter and upstream from the DNA sequence encoding the peptide orprotein of interest. RNA splice sites may be obtained from adenovirusand/or immunoglobulin genes. Also contained in the expression vectors isa polyadenylation signal located downstream of the coding sequence ofinterest. Polyadenylation signals include the early or latepolyadenylation signals from SV40 (Kaufman and Sharp, ibid.), thepolyadenylation signal from the Adenovirus 5 E1B region and the humangrowth hormone gene terminator (DeNoto et al., Nuc. Acids Res. 9:3719-3730 (1981)). The expression vectors can include a noncoding viralleader sequence, such as the Adenovirus 2 tripartite leader, locatedbetween the promoter and the RNA splice sites. Preferred vectors mayalso include enhancer sequences, such as the SV40 enhancer and the mouseμ enhancer (Gillies, Cell 33: 717-728 (1983)). Expression vectors mayalso include sequences encoding the adenovirus VA RNAs.

Cloned DNA sequences can be introduced into cultured mammalian cells by,for example, calcium phosphate-mediated transfection (Wigler et al.,Cell 14: 725 (1978); Corsaro and Pearson, Somatic Cell Genetics 7: 603(1981); Graham and Van der Eb, Virology 52: 456 (1973); which areincorporated by reference herein in their entirety). Other techniquesfor introducing cloned DNA sequences into mammalian cells may also beused, such as electropotation (Neumann et al., EMBO J. 1: 841-845(1982)) or cationic lipid-mediated transfection (Hawley-Nelson et al.,Focus 15: 73-79 (1993)) using, e.g., a 3:1 liposome formulation of2,3-dioleyloxy-N-2(sperminecarboxyamido)ethyl!-N,N-dimethyl-1-propanaminiumtrifluoroacetateand dioleolyphosphatidylethanolamine in water (Lipofectamine™ reagent,GIBCO-BRL). To identify cells that have integrated the cloned DNA, aselectable marker is generally introduced into the cells along with thegene or cDNA of interest. Examples of selectable markers for use incultured mammalian cells include genes that confer resistance to drugs,such as neomycin, hygromycin, and methotrexate. The selectable markercan be an amplifiable selectable marker. A preferred amplifiableselectable marker is the DHFR gene. Selectable markers are reviewed byThilly (Mammalian Cell Technology, Butterworth Publishers, Stoneham,Mass., which is incorporated herein by reference). The choice ofselectable markers is well within the level of ordinary skill in theart.

Selectable markers may be introduced into the cell on a separate plasmidat the same time as the gene of interest, or they may be introduced onthe same plasmid. If on the same plasmid, the selectable marker and thegene of interest may be under the control of different promoters or thesame promoter, the latter arrangement producing a dicistronic message.Constructs of this type are known in the art (for example, Levinson andSimonsen, U.S. Pat. No. 4,713,339). It may also be advantageous to addadditional DNA, known as "carrier DNA" to the mixture which isintroduced into the cells.

Transfected mammalian cells are allowed to grow for a period of time,typically 1-2 days, to begin expressing the DNA sequence(s) of interest.Drug selection is then applied to select for growth of cells that areexpressing the selectable marker in a stable fashion. For cells thathave been transfected with an amplifiable selectable marker the drugconcentration may be increased in a stepwise manner to select forincreased copy number of the cloned sequences, thereby increasingexpression levels.

Promoters, terminators and methods useful for introducing expressionvectors encoding CAP-2 or CAP-3 proteins of the present invention intoplant, avian and insect cells have been described in the art. The use ofbaculoviruses, for example, as vectors for expressing heterologous DNAsequences in insect cells has been reviewed by Atkinson et al. (Pestic.Sci. 28: 215-224 (1990)). The use of Agrobacterium rhizogenes as vectorsfor expressing genes in plant cells has been reviewed by Sinkar et al.(J. Biosci. (Bangalore) 11: 47-58 (1987)).

Host cells containing DNA constructs of the present invention are thencultured to produce CAP-2 or CAP-3 proteins. The cells are culturedaccording to standard methods in a culture medium containing nutrientsrequired for growth of the host cells. A variety of suitable media areknown in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins, minerals and growth factors.The growth medium will generally select for cells containing the DNAconstruct by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker on the DNAconstruct or co-transfected with the DNA construct.

The polypeptides of the invention, including recombinantly producedCAP-2 and CAP-3 proteins produced as described above, may be purified bytechniques well known to those of skill in the art. For example,recombinantly produced CAP-2 or CAP-3 polypeptides can be directlyexpressed or expressed as fusion proteins. The proteins can then bepurified by a combination of cell lysis (e.g., sonication) and affinitychromatography. For fusion products, subsequent digestion of the fusionprotein with an appropriate proteolytic enzyme releases the desiredpolypeptide.

The phrase "substantially purified" when referring to CAP-2 or CAP-3peptides or proteins of the present invention, means a composition whichis essentially free of other cellular components with which the CAP-2 orCAP-3 peptides or proteins are associated in their native environment.Purified protein is preferably in a homogeneous state although it can bein either a dry or aqueous solution. Purity and homogeneity aretypically determined using analytical chemistry techniques such aspolyacrylamide gel electrophoresis or high performance liquidchromatography. Generally, a substantially purified protein willcomprise more than 80% of all macromolecular species present in thepreparation. Preferably, the protein is purified to represent greaterthan 90% of all proteins present. More preferably the protein ispurified to greater than 95%, and most preferably the protein ispurified to essential homogeneity, wherein other macromolecular speciesare not detected by conventional techniques.

The CAP-2 and CAP-3 peptides and proteins of the present invention maybe purified to substantial purity by standard techniques well known inthe art, including selective precipitation with such substances asammonium sulfate; column chromatography; affinity methods, includingimmunopurification methods; and others. See, for instance, R. Scopes,Protein Purification: Principles and Practice, Springer-Verlag: New York(1982), incorporated herein by reference. For example, antibodies may beraised to the CAP-2 or CAP-3 protein as described herein. CAP-2 or CAP-3protein can be extracted from tissues or cell cultures that express theprotein and then immunoprecipitated. The CAP-2 or CAP-3 protein may thenbe further purified by standard protein chemistry techniques asdescribed above.

The CAP-2 and CAP-3 of the present invention find use as proteaseinhibitors in the purification of a wide variety of different proteins.Proteolysis is a major problem in purification of proteins. Proteolysiscan occur at all stages of purification, particularly the early stages,when more contaminating proteins are present. Because of theirwidespread distribution, serine proteases often contribute to thedegradation of proteins during purification. Several different serineprotease inhibitors have successfully been used, along with otherprotease inhibitors, during the purification of variety of differentproteins. (See, e.g., Deutscher, Meth. Enzymol., 182: 83-89 (1990)).Isolated CAP-2 or CAP-3 proteins can be used alone or in combinationwith a number of other protease inhibitors. CAP-2 or CAP-3 is used as aserine protease inhibitor at a concentration of about 10 ng-100 μg/ml,typically about 1 μg/ml.

Within one embodiment, CAP-2 or CAP-3 is covalently coupled to a solidsupport using conventional coupling chemistry. Suitable supports in thisregard include glass beads, silica-based resins, cellulosic resins,agarose beads, cross-linked agarose beads, polystyrene beads,cross-linked polyacrylamide resins and the like that are insoluble underthe conditions in which they are to be used. These supports may bemodified with reactive groups that allow attachment of proteins throughamino groups, carboxyl groups, sulphydryl groups, hydroxyl groups and/orcarbohydrate moieties. Examples of coupling chemistries include cyanogenbromide activation, N-hydroxysuccinimide activation, epoxide activation,sulphydryl activation, hydrazide activation, and carboxyl and aminoderivatives for carbodiimide coupling chemistries. In a typicalprocedure, the resin-CAP complex is packed into a column, and an aqueousmixture containing a protease is applied to the column. In general, themixture will be buffered at a pH compatible with the activity optimum ofthe protease to maximize binding of the protease to the CAP protein. Theprotease is allowed to bind to the immobilized CAP, and other componentsof the mixture pass through the column. The column is washed at the pHof the loading buffer to remove additional unbound mixture components.The bound protease can be eluted from the column with buffers thatdisrupt protein-protein interactions, such as chaotropic salts (KSCN,etc.) or high or low pH solutions. These general methods can be readilyadapted for purification of proteases or removal of proteases fromprocess streams.

The specificity of CAP-2 and CAP-3 for inhibition of different serineproteases can be determined using a variety of methods. For example,isolated biologically active CAP-2 or CAP-3 protein is added to anenzymatically active preparation of the selected serine protease, andenzyme activity is monitored to detect inhibition. For example,specificity and kinetics of inhibition for trypsin-like serine proteasescan be determined as described in Morgenstern, et al., Biochem. 33:3432-3441 (1994), incorporated herein by reference. As an additionalexample, specificity for inhibition of other proteases of interest, suchas those in the ICE family, is determined by adding CAP-2 and CAP-3 topreparations of these enzymes in vitro and monitoring changes in enzymeactivity.

Pharmacological activity of CAP-2 and CAP-3 and agonists or antagoniststhereof can also be determined in animal model systems known to those ofskill in the art. For example, CAP-2 or CAP-3 may inhibit inflammationby inhibiting the activity of ICE or other serine proteases involved inthe inflammation process. A number of in vitro and animal model systemsare used for identification of compounds with anti-inflammatoryactivity. For example, cultured vertebrate dorsal root ganglion (DRG)neurons can be transfected with an expression vector which encodes CAP-2or CAP-3 cDNA, and the inhibition of cell death or degenerationdetermined, as generally described in Gagliardini et al., Science 263:826-828 (1994), incorporated herein by reference. The effect of CAP-2 orCAP-3 and antagonists or agonists thereof can be demonstrated by othermethods, e.g., transfecting Ice, Ich-1L, or other gene which inducesprogrammed cell death or cell degeneration (e.g., ced-3) into a cellwhich is also transfected with a gene encoding CAP-2 or CAP-3, such asDRG, RAT-1 or HeLa cells, and exposing the cells to the potential CAP-2or CAP-3 agonist or antagonist compound or gene expressing saidcompound. See generally, Miura et al., Cell 75: 653-660 (1993) and Wanget al., Cell 78: 739-750 (1994), each of which is incorporated herein byreference.

The CAP-2 and CAP-3 of the present invention also have a variety of invitro diagnostic uses. For example, CAP-2 may be an endogenous inhibitorof specific trypsin-like serine proteases, and CAP-3 may be anendogenous inhibitor for members of the ICE family of proteasesincluding Ich-1. Proteases with trypsin-like specificity are involved inmany physiologically important processes, and ICE and Ich-1 playimportant roles in inflammation and apoptosis, respectively. Because ofthis, determination of CAP-2 or CAP-3 in biological samples can beuseful in medicine.

Levels of CAP-2 or CAP-3 protein can be determined, for example, bymeans of a variety of different immunoassay procedures. Antibodies, bothpolyclonal and monoclonal, can be produced to CAP-2 and CAP-3 proteinsand polypeptide fragments thereof according to general procedures setforth in Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Pubs., New York (1988), incorporated herein by reference.Antibodies raised to a CAP-2 immunogen, e.g., that having the amino acidsequence depicted in Seq. ID No. 2, can be selected via screeningprocedures to be specifically immunoreactive with CAP-2 proteins and notwith other proteins such as CAP-1 protein and CAP-3 protein. Similarly,antibodies raised to CAP-3 immunogen, e.g., that having the amino acidsequence depicted in Seq. ID No. 4, can be selected to be specificallyimmunoreactive with CAP-3 proteins and not with other proteins such asCAP-1 protein and CAP-2 protein. A variety of immunoassay formats may beused to select antibodies specifically immunoreactive with a particularprotein. For example, solid-phase ELISA immunoassays are routinely usedto select monoclonal antibodies specifically immunoreactive with aprotein. See Harlow and Lane, supra, for a description of immunoassayformats and conditions that can be used to determine specificimmunoreactivity.

Diagnostic immunoassays for CAP-2 or CAP-3 in a biological sample can beperformed in a variety of different formats known to those of skill inthe art and described in, e.g., Harlow and Lane, supra; Basic andClinical Immunology 7th Edition (D. Stites and A. Terr ed.) 1991; EnzymeImmunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Fla. (1980); and"Practice and Theory of Enzyme Immunoassays," P. Tijssen, LaboratoryTechniques in Biochemistry and Molecular Biology, Elsevier SciencePublishers B. V. Amsterdam (1985), each of which is incorporated hereinby reference. By biological sample is meant to include body fluids andtissue specimens, that is, any sample derived from or containing cells,cell components or cell products, including, but not limited to, cellculture supernatants, cell lysates, cleared cell lysates, cell extracts,tissue extracts, blood, plasma, serum, and fractions thereof.

Expression of mRNA encoding CAP-2 or CAP-3 proteins can be detected byvarious procedure involving nucleic acid hybridization. A variety ofnucleic acid hybridization formats are known to those skilled in theart. For example, common formats include sandwich assays and competitionor displacement assays. Hybridization techniques are generally describedin "Nucleic Acid Hybridization, A Practical Approach," Ed. Hames, B. D.and Higgins, S. J., IRL Press, 1985; Gall and Pardue (1969), Proc. Natl.Acad. Sci., U.S.A., 63:378-383; and John et al., (1969) Nature,223:582-587, and in Sambrook, et al. Hybridization techniques can bealso used in methods such as restriction fragment length polymorphism(RFLP) analysis to detect the presence of genetic alterations in nucleicacids encoding CAP-2 and CAP-3 (see Sambrook, et al., supra).

The CAP-2 and CAP-3 protein compositions of the present invention areuseful in treatment and prevention of a variety of diseases, e.g.,inflammatory diseases. The CAP-2 or CAP-3 composition is used to preventneuronal degeneration. Inhibition of βIL-1 maturation indicates thesubject composition can be used to treat Alzheimer's disease, arthritis,septic shock, head injury, and other inflammatory responses.

Pharmaceutical compositions of the invention are suitable for use in avariety of drug delivery systems. Pharmaceutically acceptable carriersand formulations for use in the present invention are found inRemington's Pharmaceutical Sciences, Mack Publishing Company,Philadelphia, Pa., 17th ed. (1985), which is incorporated herein byreference. For a brief review of methods for drug delivery, see Langer,Science 249:1527-1533 (1990), which is incorporated herein by reference.

In preparing pharmaceutical compositions of the present invention, itmay be desirable to modify the compositions of the present invention toalter their pharmacokinetics and biodistribution. For a generaldiscussion of pharmacokinetics, see Remington's Pharmaceutical Sciences,supra, Chapters 37-39. A number of methods for altering pharmacokineticsand biodistribution are known to one of ordinary skill in the art (See,e.g., Langer, supra). Examples of such methods include protection of thecomplexes in vesicles composed of substances such as proteins, lipids(for example, liposomes), carbohydrates, or synthetic polymers. Forexample, the complexes of the present invention may be incorporated intoliposomes in order to enhance their pharmacokinetics and biodistributioncharacteristics. A variety of methods are available for preparingliposomes, as described in, e.g., Szoka et al., Ann. Rev. Biophys.Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728 and 4,837,028,all of which are incorporated herein by reference.

The CAP-2 and CAP-3 proteins of the present invention can be used inpharmaceutical compositions that are useful for administration tomammals, including selective pharmaceutical compositions of theinvention are intended for parenteral, topical, oral or localadministration. For example, the pharmaceutical compositions can beadministered parenterally, e.g., intravenously, subcutaneously,intradermally, or intramuscularly. The invention provides compositionsthat comprise a solution of the agents described above dissolved orsuspended in an acceptable carrier, preferably an aqueous carrier. Avariety of pharmaceutically acceptable aqueous carriers may be used,e.g., water, buffered water, 0.4% saline, 0.3% glycine, hyaluronic acidand the like. These compositions may be sterilized by conventional, wellknown sterilization techniques, or may be sterile filtered. Theresulting aqueous solutions may be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterilesolution prior to administration. The compositions may contain aspharmaceutically acceptable carriers, substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, triethanolamineoleate, etc.

For solid compositions, conventional nontoxic pharmaceuticallyacceptable carriers may be used which include, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, talcum, cellulose, glucose, sucrose, magnesiumcarbonate, and the like. For oral administration, a pharmaceuticallyacceptable nontoxic composition is formed by incorporating any of thenormally employed excipients, such as those carriers previously listed,and generally 10-95% of active ingredient and more preferably at aconcentration of 25%-75%.

For aerosol administration, the pharmaceutical compositions containingthe CAP-2 or CAP-3 proteins or peptide fragments thereof are preferablysupplied in finely divided form along with a surfactant and propellantas pharmaceutically acceptable carriers. The surfactant must, of course,be nontoxic, and preferably soluble in the propellant. Representative ofsuch agents are the esters or partial esters of fatty acids containingfrom 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic,stearic, linoleic, linolenic, olesteric and oleic acids with analiphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, suchas mixed or natural glycerides, may be employed. A carrier can also beincluded, as desired, as with, e.g., lecithin for intranasal delivery.

The pharmaceutical compositions of the invention can be administered ina variety of unit dosage forms depending upon the method ofadministration. For example, unit dosage forms suitable for oraladministration include powder, tablets, pills, and capsules. Theeffective amount of the CAP-2 or CAP-3 protein in a pharmaceuticalcomposition will depend on, e.g., the protein composition, the manner ofadministration, the weight and general state of health of the patient,the severity of the disease being treated and the judgment of theprescribing physician. Dosages, formulations and administrationschedules may vary in these patients compared to normal individuals. Ingeneral, dosages range from about 100 μg to about 500 mg or more, withdosages of from about 250 μg to about 50 mg being more commonly used. Itmust be kept in mind that the materials of the present invention maygenerally be employed in serious disease or injury states, and in suchcases it is possible and may be felt desirable by the treating physicianto administer substantial excesses of these CAP-2 or CAP-3 compositions.

The following Examples are offered by way of illustration of the presentinvention, not limitation.

EXAMPLES Example 1

Isolation of cDNA molecules encoding human

CAP-2 and CAP-3 proteins

A. Generation of a nucleic acid probe for screening a human cDNA library

To isolate cDNA molecules encoding CAP-1 and CAP-2 proteins, a humanplacenta λgt11 cDNA library was screened using an antisense 209 basepair PCR-generated ³² P-labeled probe corresponding to codons encodingresidues 67-149 of the CAP-1 protein. (See Morgenstern et al. (1994)supra.) This probe was generated in a series of PCR reactions, asdescribed below.

In the first PCR reaction, a human placenta cDNA library (Cat. #HL1075b, ClonTech, Palo Alto, Calif.) was amplified using oligonucleotideZC6657 (Seq. ID No. 5) and oligonucleotide ZC6658 (Seq. ID No. 6). Theseoligonucleotides are degenerate primers based on a peptide sequenceisolated from the CAP-1 protein. (See Morgenstern et al. (1994) supra.)The PCR reaction was generated using Ampliwax™ (Roche Molecular Systems,Branchburg, N.J.) and a "hot start" technique that prevents falsepriming by adding the enzyme to the PCR reaction at an elevatedtemperature. More specifically, PCR was performed by mixing 21 μl H20, 8μl dNTP (2.5 mM each dNTP), 8 μl of 20 pM/μl oligonucleotide ZC6657, 8μl of 20 pM/μl oligonucleotide ZC6658, and 5 μl of 10× GeneAmp® PCRbuffer. Ampliwax™ was added, and the reaction mixture was heated at 80°C. for 5 minutes and then at 35° C. for 2 minutes. After the waxhardened on top of the reaction mixture, 2 μl of the human placenta cDNAlibrary was diluted into 42 μl of water and boiled 5-10 minutes. Thediluted cDNA library was added above the cooled wax with 1 μl AmpliTaq®(5 units/μl; Roche) and 5 μl of 10× GeneAmp® PCR buffer (Roche). Thereaction mixture was incubated for 30 cycles of the followingtemperatures: 95° C. for 30 seconds; 48° C. for 30 seconds; and 72° C.for 1 minute. This was followed by a 7 minute incubation at 72° C.

The product of the first PCR reaction was then used as a template in asecond PCR reaction to generate a product of approximately 270 bp. Areaction mixture was prepared by combining 21 μl H₂ O, 8 μl dNTPs (2.5mM each), 8 μl of the ZC6657 oligonucleotide (20 pM/μl, 8 μl of theZC6658 oligonucleotide (20 pM/μl), and 5 μl of 10× GeneAmp® PCR buffer.Ampliwax™ was added, and the reaction mixture was heated at 80° C. for 5minutes, then at 35° C. for 2 minutes. After the wax hardened, 43 μl H₂O, 1 μl AmpliTaq® (5 units/μl), 5 μl 10× GeneAmp® PCR buffer, and 1 μltemplate DNA were added above the cooled wax. The reaction mixture wasthen incubated for 30 cycles at the following temperatures: 95° C. for30 seconds; 48° C. for 30 seconds; and 72° C. for 1 minute. This wasfollowed by a 7 minute incubation at 72° C. The final PCR product wasdigested with EcoRI and ligated into the EcoRI digested vector ZEM228CC, described below. The resulting construct was designated "clone 10CAP-Zem228CC".

The vector Zem228CC was prepared from plasmid Zem228, a pUC18-basedexpression vector containing a unique Bam HI site for insertion ofcloned DNA between the mouse metallothionein-1 promoter and SV40transcription terminator and an expression unit containing the SV40early promoter, neomycin resistance gene, and SV40 terminator. Zem228was deposited with American Type Culture Collection, 12301 ParklawnDrive, Rockville, Md. on Sep. 28, 1993 as an E. coli HB101 transformant.It has been assigned Accession Number 69446. Plasmid Zem228 was modifiedto delete the two Eco RI sites by partial digestion with Eco RI,blunting with DNA polymerase I (Klenow fragment) in the presence ofdNTPs, and re-ligation. Digestion of the resulting plasmid with Bam HIfollowed by ligation of the linearized plasmid with Bam HI-Eco RIadapters resulted in a unique Eco RI cloning site. The resultant plasmidwas designated Zem228R. The Sst I site between SV40 promoter and themouse metallothionein-1 promoter was destroyed by linearizing Zem228Rwith Sst I, blunting the adhesive ends with T4 DNA polymerase in thepresence of dNTPs and religating the linearized, blunt-ended fragment. Aplasmid in which the Sst I site was destroyed was designed Zem228Ra.

To facilitate directional insertion of cDNA fragments into the vector,an adapter was synthesized which contained a 5' Eco RI adhesive end, aninternal Sst I site and a 3' Eco RI adhesive end that does notregenerate an Eco RI site upon ligation with an Eco RI adhesive end.Plasmid Zem228Ra was linearized by digestion with Eco RI, and thelinearized plasmid was treated with calf alkaline phosphatase to preventrecircularization. The linearized plasmid was ligated with kinasedoligonucleotides ZC3169 and ZC3168 (see Table). A plasmid containinginserted adapter was designated Zem228C.

To improve the ability to achieve Eco RI+Sst I cleavage of the Zem228Cvector, an oligonucleotide adapter was synthesized that contained aninternal Eco RI site flanked by Eco RI adhesive ends that do notregenerate Eco RI sites upon ligation with Eco RI adhesive ends.Oligonucleotides ZC1773 and ZC1774 (Table) were kinased and annealed toform the adapter. Plasmid Zem228C was linearized by digestion with EcoRI, and the linearized vector and kinased adapter were ligated. Aplasmid containing the adapter was confirmed and sequenced. Sequenceanalysis revealed that the plasmid contained a 30 bp DNA insert betweenthe new Eco RI site and the downstream Sst I site. Since an Eco RI+Sst Icleavage of the vector prior to the insertion of a cDNA sequence removesthe additional DNA sequence, the inserted DNA was not removed. Theplasmid was designated Zem228CC.

                                      TABLE                                       __________________________________________________________________________    ZC1773:                                                                            AATTAGGGAG                                                                            ACCGGAATTC                                                                            TGTGCTCTGT                                                                            CAA                                                                              (SEQ. ID No. 13)                              ZC1774:                                                                            AATTTTGACA                                                                            GAGCACAGAA                                                                            TTCCGGTCTC                                                                            CTT                                                                              (SEQ. ID No. 14)                              ZC3168:                                                                            AATTGAGCTC                                                                            G (SEQ. ID No. 15)                                               ZC3169:                                                                            AATTCGAGCT                                                                            C (SEQ. ID No. 16)                                               __________________________________________________________________________

The plasmid clone 10 CAP-Zem 228CC was used as a template to generate aprobe for screening a human placenta cDNA library for nucleic acidsequences structurally related to CAP-1. A PCR reaction was carried outusing the plasmid template and the oligonucleotide primers ZC6770 (Seq.ID No. 7) and ZC6771 (Seq. ID No. 8). PCR was performed in a 50 μlreaction volume with approximately 10 ng plasmid DNA, 40 pmoles ofoligonucleotide ZC6770, 40 pmoles of oligonucleotide ZC6771, 5 μl of 10×GeneAmp® PCR buffer, 5 μl dNPS (2.5 mM each dNTP), and 0.25 μl AmpliTaq™ DNA polymerase (5 Units/μl). The reaction mixture was incubated for 35cycles at the following temperatures: 94° C. for 1 minute; 55° C. for 1minute; and 72° C. for 1.5 minutes. This was followed by a 7 minuteincubation at 72° C. The 210 bp product was labeled with ³² P using acommercially available kit (Multi-prime kit, Amersham Corporation,Arlington Heights, Ill., U.S.A.).

B. Library screening and isolation and characterization of cDNA clonesencoding human CAP-2 and CAP-3 proteins

33,000 phage from the human placenta cDNA library were plated on each of23 plates to obtain 760,000 independent phage. Filter lifts were madefrom each plate using ICN BioTrans nylon membranes. Filters wereprehybridized in 5×SSPE (20× SSPE is 175.3 g NaCl, 27.6 g NaH2PO4.H₂ O,7.4 g EDTA, NaOH added to pH7.4 and water to 1 liter), 5×Denhardt's,0.5% SDS, 100 μg/ml salmon sperm DNA at 65° C. for 6 hours. Filters werethen hybridized in the above mixture with 1×10⁶ cpm/ml of labeled CAPprobe at 65° C. overnight. The filters were washed 3 times for 40minutes each at room temperature in 0.2×SSC, 0.1% SDS, followed by 1wash for 40 minutes at 65° C. Filters were exposed to X-ray filmovernight. Two types of positive spots were observed: those which wereextremely intense and corresponded to CAP cDNA, and those that gave muchweaker signals.

For two of the weak signals, the corresponding phage wereplaque-purified by additional rounds of hybridization and isolationusing nitrocellulose filters. In some cases, filters were washed priorto hybridization in 5×SSC, 0.1% SDS at 65° C. for I hour; hybridizationwas carried out in 5×SSC at 56° C. overnight; and filters were washed at58° C. in 2×SSC, 0.1% SDS. Two plaques were purified by this procedure.The two purified plaques were designated H2-2-11 and H3-1-11.

cDNA inserts were isolated from each purified plaque by PCR, using phageas a template and amplifying with oligonucleotide primers ZC2682 (Seq.ID No. 9) and ZC2683 (Seq. ID No.10), which anneal 5' and 3' to theEcoRI cloning site of λgt11. A 50 μl PCR reaction volume was used with 1μl eluted phage, 40 pmoles of oligonucleotide ZC2682, 40 pmoles ofoligonucleotide ZC2683, 5 μl 10× PFU buffer (Stratagene Cloning Systems,Stratagene, San Diego, Calif., U.S.A.), 5 μl dNTPs (2.5 mM each dNTP;Roche), and 1 μl PFU polymerase, 2.5 Units/μl (Stratagene, San Diego,Calif., U.S.A.). The reaction mixture was incubated for 35 cycles at thefollowing temperatures: 94° C. for 1 minute; 60° C. for i minute; and72° C. for 2 minutes. This was followed by a 7 minute incubation at 72°C. The resulting PCR products were designated H2-2-11 and H3-1-11.

The H2-2-11 and H3-1-11PCR products were digested with EcoRI andindividually ligated into the EcoRI site of pUC19. The H2-2-11 sequencewas isolated as a 1.4 kb fragment. H3-1-11 contained an internal EcoRIsite in the cDNA, resulting in a 1.1 kb fragment and a 0.3 kb fragmentupon digestion with EcoRI. Both cDNAs were sequenced and determined tobe related to CAP-1 and other intracellular members of the serpin familyof protease inhibitors. The nucleotide sequences and the deduced aminoacid sequences of the inserts in clones H3-1-11 and H2-2-11 are shown inSEQ ID NO: 1-4. For both cDNAs, the active site region was sequencedfrom 2 independent PCR products to check for any PCR-generated errors.

The 5'-regions of both cDNAs contain a Kozak consensus sequence betweennucleotide bases 110-119 of H2-2-11 (SEQ. ID. No. 3) and 88-97 ofH3-1-11 (SEQ. ID. No. 1) that includes an in frame initiation codon. Asecond Kozak sequence also exists 117 nucleotide bases downstream of thefirst initiation codon and includes the codon for Met⁴¹ of bothproteins. The 3'-untranslated region of the H3-1-11 cDNA contains anAATAAA consensus sequence located 99 nucleotide bases downstream of thetermination codon for nascent mRNA cleavage and polyadenylation.However, a polyadenylation consensus sequence was not found in the3'-untranslated region of the H2-2-11 cDNA after sequencing 151nucleotides downstream from the translational termination codon.

Alignment of the deduced primary structure of CAP-2 and CAP-3 with theamino acid sequences of CAP-1 and other human members of the ovalbuminserpin family identified the putative reactive center P₁ -P₁ ' residuesof CAP-2 as Arg³³⁹ -Cys³⁴⁰, respectively, which are identical to theCAP-1 reactive center residues. However, the regions flanking the P₁ -P₁' residues in CAP-1 and CAP-2 are highly divergent. The P₂ -P₆ residuesof CAP-1 and CAP-2 show no identity while Arg³⁴² in the P₃ ' positionwas conserved in both serpins. Residues in the vicinity of P₁ have beenpreviously shown to influence both proteinase target specificity and theinhibitory potency of several serpins. Carrell et al., Nature 353:576-578 (1991). Thus, this indicates that CAP-2 interacts with theactive sites of distinct cognate proteinases that have trypsin-likesubstrate specificity. In contrast, alignment of the CAP-3 amino acidsequence identified the putative P₁ -P₁ ' residues at Glu³⁴⁰ -Cys³⁴¹,respectively.

The identification of an acidic P₁ residue in the CAP-3 reactive centeris unique in the mammalian serpin superfamily. The only other serpinidentified with an acidic P₁ residue in the reactive center is encodedby the crmA gene of the cowpox virus, which has been previouslydemonstrated to have a reactive center containing an Asp-Cys in the P₁-P₁ ' positions, respectively. Furthermore, the crmA protein shows thegreatest degree of amino acid sequence identity to the mammalianintracellular serpins of the ovalbumin family. By employing the NBRFprogram ALIGN, the crmA protein was found to share about 37% identitywith CAP-2 and CAP-3. The CAP-3 reactive center domain shares about 54%of structurally conserved residues found in the reactive center domainof the crmA protein, including a conserved Asp to Glu switch in the P₁-specificity site. Moreover, the cytoplasmic antiproteinases have aunique Cys-residue conserved in the P₁ ' position and found only in thecorresponding position of the crmA serpin reactive center.

The crmA protein functions as a specific inhibitor of ICE whichrepresents a prototype of a larger family of ICE-like homologs. The ICEfamily of cysteine proteinases have been linked to both the negative andpositive regulation of apoptosis. A human homolog of ICE has beenidentified and designated as Ich-1. In contrast to ICE, Ich-1-mediatedeffects on apoptosis of Rat-1 cells is only partially blocked by eithermicroinjected or coexpressed crmA protein. These findings suggest thatIch-1 and the crmA serpin interact weakly and further suggests thatIch-1 and ICE have distinct but overlapping substrate specificities.

Serp2-2, the cDNA encoding CAP-3 (H2-2-11), inserted into pUC19 as anEcoRI fragment; Serp3-1a (H3-1-11), the 5' EcoRI fragment of the CAP-2clone inserted into pUC19; and Serp3-1b (H3-1-11), the 3' EcoRI fragmentof the CAP-2 clone inserted into pUC19, were deposited with AmericanType Culture Collection, 12301 Parklawn Drive, Rockville, Md. on Oct. 6,1994 as E. coli DH10b transformants and assigned Accession Numbers69699, 69700, and 69701, respectively.

Example 2 Northern blot analysis of human tissues for expression ofCAP-2 and CAP-3 mRNA

To identify transcripts that encode CAP-2 and CAP-3 and determine theirhuman tissue distribution, radiolabeled oligonucleotide probescorresponding to the reactive centers of these serpins were used toprobe immobilized poly(A)+ mRNA by Northern analysis. A blot ofpoly(A)-containing mRNA from human tissues used a multiple tissue blotfrom CLONTECH Laboratories, Palo Alto, Calif., U.S.A. The multipletissue blot was probed with a 39-mer oligonucleotide corresponding tothe reactive centers of the CAP homologs. The oligonucleotide used forCAP-2 has the following sequence:5'-CTCCATTCTGCTGCACCGGGAATTCCTGACCACAGCAGT-3' (Sequence ID No. 11). Theoligonucleotide used for CAP-3 had the following sequence:5'-AGATTCCATGCAGCACTCTGCAACTACAAAGCAGCTCGA-3' (Seq. ID No. 12). Theoligonucleotide probes were 5'-labeled with ³² Pγ!ATP (Dupont/NEN,Boston, Mass., U.S.A.) using T4 polynucleotide kinase (PromegaCorporation, Madison, Wis., U.S.A.) to yield a specific activity ofabout 1-2×10⁸ cpm/μg. Hybridization was performed at 55° C. in 5× SSPE,2× Denhardt's, 0.5 SDS, 100 μg/ml salmon sperm DNA. The blots werewashed at 57° C. in 2× SSC, 0.1% SDS and exposed to autoradiography.

The results indicated that two mRNA species of 3.4 kbp and 4.4 kbp weredetected with the CAP-3 reactive center antisense probe. Both mRNAspecies encoding the CAP-3 reactive center were detected at the highestlevels in placenta and lung and to a much lesser degree in all tissuesexamined. In addition, two minor mRNA species of approximately 7.5-8.0kbp were also detected in these tissues. The hybridization of theNorthern blots was of sufficient stringency to preclude hybridization ofinexact nucleotide matches.

The above examples are provided to illustrate the invention but not tolimit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, patents, and patent applicationscited herein are hereby incorporated by reference.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 16                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1425 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 92..1213                                                        (D) OTHER INFORMATION: /product="CYTOPLASMIC                                  ANTIPROTEINASE-2 PROTEIN"                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GAATTCGGAGCATCTACAAAGGAGGAATAGTCAAAGCAGCAGCGGCGGCGGCGGCGGCGG60                CAGCAGCAGCAGCAGCAGGAGACCTTCTCTGATGGATGACCTCTGTGAAGCA112                       MetAspAspLeuCysGluAla                                                         15                                                                            AATGGCACTTTTGCCATCAGCTTATTTAAAATATTGGGGGAAGAGGAC160                           AsnGlyThrPheAlaIleSerLeuPheLysIleLeuGlyGluGluAsp                              101520                                                                        AACTCAAGAAACGTATTCTTCTCTCCCATGAGCATCTCCTCTGCCCTG208                           AsnSerArgAsnValPhePheSerProMetSerIleSerSerAlaLeu                              253035                                                                        GCCATGGTCTTCATGGGGGCAAAGGGAAGCACTGCAGCCCAGATGTCC256                           AlaMetValPheMetGlyAlaLysGlySerThrAlaAlaGlnMetSer                              40455055                                                                      CAGGCACTTTGTTTATACAAAGACGGAGATATTCACCGAGGTTTCCAG304                           GlnAlaLeuCysLeuTyrLysAspGlyAspIleHisArgGlyPheGln                              606570                                                                        TCACTTCTCAGTGAAGTTAACAGAACTGGCACTCAGTACTTGCTTAGA352                           SerLeuLeuSerGluValAsnArgThrGlyThrGlnTyrLeuLeuArg                              758085                                                                        ACTGCCAACAGACTCTTTGGAGAAAAGACGTGTGATTTCCTTCCAGAC400                           ThrAlaAsnArgLeuPheGlyGluLysThrCysAspPheLeuProAsp                              9095100                                                                       TTTAAAGAATACTGTCAGAAGTTCTATCAGGCAGAGCTGGAGGAGTTG448                           PheLysGluTyrCysGlnLysPheTyrGlnAlaGluLeuGluGluLeu                              105110115                                                                     TCCTTTGCTGAAGACACTGAAGAGTGCAGGAAGCATATAAATGACTGG496                           SerPheAlaGluAspThrGluGluCysArgLysHisIleAsnAspTrp                              120125130135                                                                  GTGGCAGAGAAGACTGAAGGTAAGATTTCAGAGGTACTGGATGCTGGG544                           ValAlaGluLysThrGluGlyLysIleSerGluValLeuAspAlaGly                              140145150                                                                     ACAGTCGATCCCCTGACAAAGCTAGTCCTTGTGAATGCCATTTATTTC592                           ThrValAspProLeuThrLysLeuValLeuValAsnAlaIleTyrPhe                              155160165                                                                     AAGGGAAAGTGGAATGAGCAATTTGACAGAAAGTACACAAGGGGAATG640                           LysGlyLysTrpAsnGluGlnPheAspArgLysTyrThrArgGlyMet                              170175180                                                                     CTCTTTAAAACCAACGAGGAAAAAAAGACAGTGCAGATGATGTTTAAG688                           LeuPheLysThrAsnGluGluLysLysThrValGlnMetMetPheLys                              185190195                                                                     GAAGCTAAGTTTAAAATGGGGTATGCGGATGAGGTACACACCCAGGTC736                           GluAlaLysPheLysMetGlyTyrAlaAspGluValHisThrGlnVal                              200205210215                                                                  CTGGAGCTGCCCTATGTGGAAGAGGAGCTGAGCATGGTCATTCTGCTT784                           LeuGluLeuProTyrValGluGluGluLeuSerMetValIleLeuLeu                              220225230                                                                     CCCGATGACAACACGGACCTCGCCGTGGTGGAAAAAGCACTTACATAT832                           ProAspAspAsnThrAspLeuAlaValValGluLysAlaLeuThrTyr                              235240245                                                                     GAGAAATTCAAAGCCTGGACAAATTCAGAAAAGTTGACAAAAAGTAAG880                           GluLysPheLysAlaTrpThrAsnSerGluLysLeuThrLysSerLys                              250255260                                                                     GTTCAAGTTTTCCTTCCCAGATTAAAGCTGGAGGAGAGTTATGACTTG928                           ValGlnValPheLeuProArgLeuLysLeuGluGluSerTyrAspLeu                              265270275                                                                     GAGCCTTTCCTTCGAAGATTAGGAATGATCGATGCTTTTGACGAAGCC976                           GluProPheLeuArgArgLeuGlyMetIleAspAlaPheAspGluAla                              280285290295                                                                  AAGGCAGACTTTTCTGGAATGTCAACTGAGAAGAATGTGCCTCTGTCC1024                          LysAlaAspPheSerGlyMetSerThrGluLysAsnValProLeuSer                              300305310                                                                     AAGGTTGCCCACAAGTGCTTCGTGGAGGTCAATGAGGAAGGCACAGAG1072                          LysValAlaHisLysCysPheValGluValAsnGluGluGlyThrGlu                              315320325                                                                     GCTGCCGCAGCCACTGCTGTGGTCAGGAATTCCCGGTGCAGCAGAATG1120                          AlaAlaAlaAlaThrAlaValValArgAsnSerArgCysSerArgMet                              330335340                                                                     GAGCCAAGATTCTGTGCAGACCACCCTTTTCTTTTCTTCATCAGGCGC1168                          GluProArgPheCysAlaAspHisProPheLeuPhePheIleArgArg                              345350355                                                                     CACAAAACCAACTGCATCTTGTTCTGTGGCAGGTTCTCTTCTCCG1213                             HisLysThrAsnCysIleLeuPheCysGlyArgPheSerSerPro                                 360365370                                                                     TAAAGAGGAGCAATTGCTGTACATACCCTCCTTTCCTTCTACCTATCTTGCCTTAATTAA1273              CATTCCCTGTGACCTAGTTGGTGCAGTGGCTTGAATGCCAAAATAAAGCGTGTGCACTGG1333              AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACCGAATTCCGCCGA1393              TACTGACGGGCTCCAGGAGTCAATCACTAGTG1425                                          (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 374 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetAspAspLeuCysGluAlaAsnGlyThrPheAlaIleSerLeuPhe                              151015                                                                        LysIleLeuGlyGluGluAspAsnSerArgAsnValPhePheSerPro                              202530                                                                        MetSerIleSerSerAlaLeuAlaMetValPheMetGlyAlaLysGly                              354045                                                                        SerThrAlaAlaGlnMetSerGlnAlaLeuCysLeuTyrLysAspGly                              505560                                                                        AspIleHisArgGlyPheGlnSerLeuLeuSerGluValAsnArgThr                              65707580                                                                      GlyThrGlnTyrLeuLeuArgThrAlaAsnArgLeuPheGlyGluLys                              859095                                                                        ThrCysAspPheLeuProAspPheLysGluTyrCysGlnLysPheTyr                              100105110                                                                     GlnAlaGluLeuGluGluLeuSerPheAlaGluAspThrGluGluCys                              115120125                                                                     ArgLysHisIleAsnAspTrpValAlaGluLysThrGluGlyLysIle                              130135140                                                                     SerGluValLeuAspAlaGlyThrValAspProLeuThrLysLeuVal                              145150155160                                                                  LeuValAsnAlaIleTyrPheLysGlyLysTrpAsnGluGlnPheAsp                              165170175                                                                     ArgLysTyrThrArgGlyMetLeuPheLysThrAsnGluGluLysLys                              180185190                                                                     ThrValGlnMetMetPheLysGluAlaLysPheLysMetGlyTyrAla                              195200205                                                                     AspGluValHisThrGlnValLeuGluLeuProTyrValGluGluGlu                              210215220                                                                     LeuSerMetValIleLeuLeuProAspAspAsnThrAspLeuAlaVal                              225230235240                                                                  ValGluLysAlaLeuThrTyrGluLysPheLysAlaTrpThrAsnSer                              245250255                                                                     GluLysLeuThrLysSerLysValGlnValPheLeuProArgLeuLys                              260265270                                                                     LeuGluGluSerTyrAspLeuGluProPheLeuArgArgLeuGlyMet                              275280285                                                                     IleAspAlaPheAspGluAlaLysAlaAspPheSerGlyMetSerThr                              290295300                                                                     GluLysAsnValProLeuSerLysValAlaHisLysCysPheValGlu                              305310315320                                                                  ValAsnGluGluGlyThrGluAlaAlaAlaAlaThrAlaValValArg                              325330335                                                                     AsnSerArgCysSerArgMetGluProArgPheCysAlaAspHisPro                              340345350                                                                     PheLeuPhePheIleArgArgHisLysThrAsnCysIleLeuPheCys                              355360365                                                                     GlyArgPheSerSerPro                                                            370                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1393 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 112..1239                                                       (D) OTHER INFORMATION: /product="CYTOPLASMIC                                  ANTIPROTEINASE-3 PROTEIN"                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GAATTCGGCGCAGCAGCAGGGCCGGGTCCTGCGCCTCGGGGGTCGGCGTCCAGGCTCGGA60                GCGCGGCACGGAGACGGCGGCAGCGCTGGACTAGGTGGCAGGCCCTGCATCATGGAA117                  MetGlu                                                                        ACTCTTTCTAATGCAAGTGGTACTTTTGCCATACGCCTTTTAAAGATA165                           ThrLeuSerAsnAlaSerGlyThrPheAlaIleArgLeuLeuLysIle                              51015                                                                         CTGTGTCAAGATAACCCTTCGCACAACGTGTTCTGTTCTCCTGTGAGC213                           LeuCysGlnAspAsnProSerHisAsnValPheCysSerProValSer                              202530                                                                        ATCTCCTCTGCCCTGGCCATGGTTCTCCTAGGGGCAAAGGGAAACACC261                           IleSerSerAlaLeuAlaMetValLeuLeuGlyAlaLysGlyAsnThr                              35404550                                                                      GCAACCCAGATGGCCCAGGCACTGTCTTTAAACACAGAGGAAGACATT309                           AlaThrGlnMetAlaGlnAlaLeuSerLeuAsnThrGluGluAspIle                              556065                                                                        CATCGGGCTTTCCAGTCGCTTCTCACTGAAGTGAACAAGGCTGGCACA357                           HisArgAlaPheGlnSerLeuLeuThrGluValAsnLysAlaGlyThr                              707580                                                                        CAGTACCTGCTGAGAACGGCCAACAGGCTCTTTGGAGAGAAAACTTGT405                           GlnTyrLeuLeuArgThrAlaAsnArgLeuPheGlyGluLysThrCys                              859095                                                                        CAGTTCCTCTCAACGTTTAAGGAATCCTGTCTTCAATTCTACCATGCT453                           GlnPheLeuSerThrPheLysGluSerCysLeuGlnPheTyrHisAla                              100105110                                                                     GAGCTGAAGGAGCTTTCCTTTATCAGAGCTGCAGAAGAGTCCAGGAAA501                           GluLeuLysGluLeuSerPheIleArgAlaAlaGluGluSerArgLys                              115120125130                                                                  CACATCAACACCTGGGTCTCAAAAAAGACCGAAGGTAAAATTGAAGAG549                           HisIleAsnThrTrpValSerLysLysThrGluGlyLysIleGluGlu                              135140145                                                                     TTGTTGCCGGGTAGCTCAATTGATGCAGAAACCAGGCTGGTTCTTGTC597                           LeuLeuProGlySerSerIleAspAlaGluThrArgLeuValLeuVal                              150155160                                                                     AATGCCATCTACTTCAAAGGAAAGTGGAATGAACCGTTTGACGAAACA645                           AsnAlaIleTyrPheLysGlyLysTrpAsnGluProPheAspGluThr                              165170175                                                                     TACACAAGGGAAATGCCCTTTAAAATAAACCAGGAGGAGCAAAGGCCA693                           TyrThrArgGluMetProPheLysIleAsnGlnGluGluGlnArgPro                              180185190                                                                     GTGCAGATGATGTATCAGGAGGCCACGTTTAAGCTCGCCCACGTGGGC741                           ValGlnMetMetTyrGlnGluAlaThrPheLysLeuAlaHisValGly                              195200205210                                                                  GAGGTGCGCGCGCAGCTGCTGGAGCTGCCCTACGCCAGGAAGGAGCTG789                           GluValArgAlaGlnLeuLeuGluLeuProTyrAlaArgLysGluLeu                              215220225                                                                     AGCCTGCTGGTGCTGCTGCCTGACGACGGCGTGGAGCTCAGCACGGTG837                           SerLeuLeuValLeuLeuProAspAspGlyValGluLeuSerThrVal                              230235240                                                                     GAAAAAAGTCTCACTTTTGAGAAACTCACAGCCTGGACCAAGCCAGAC885                           GluLysSerLeuThrPheGluLysLeuThrAlaTrpThrLysProAsp                              245250255                                                                     TGTATGAAGAGTACTGAGGTTGAAGTTCTCCTTCCAAAATTTAAACTA933                           CysMetLysSerThrGluValGluValLeuLeuProLysPheLysLeu                              260265270                                                                     CAAGAGGATTATGACATGGAATCTGTGCTTCGGCATTTGGGAATTGTT981                           GlnGluAspTyrAspMetGluSerValLeuArgHisLeuGlyIleVal                              275280285290                                                                  GATGCCTTCCAACAGGGCAAGGCTGACTTGTCGGCAATGTCAGCGGAG1029                          AspAlaPheGlnGlnGlyLysAlaAspLeuSerAlaMetSerAlaGlu                              295300305                                                                     AGAGACCTGTGTCTGTCCAAGTTCGTGCACAAGAGTTTTGTGGAGGTG1077                          ArgAspLeuCysLeuSerLysPheValHisLysSerPheValGluVal                              310315320                                                                     AATGAAGAAGGCACCGAGGCAGCGGCAGCGTCGAGCTGCTTTGTAGTT1125                          AsnGluGluGlyThrGluAlaAlaAlaAlaSerSerCysPheValVal                              325330335                                                                     GCAGAGTGCTGCATGGAATCTGGCCCCAGGTTCTGTGCTGACCACCCT1173                          AlaGluCysCysMetGluSerGlyProArgPheCysAlaAspHisPro                              340345350                                                                     TTCCTTTTCTTCATCAGGCACAACAGAGCCAACAGCATTCTGTTCTGT1221                          PheLeuPhePheIleArgHisAsnArgAlaAsnSerIleLeuPheCys                              355360365370                                                                  GGCAGGTTCTCATCGCCATAAAGGGTGCACTTACCGTGCACTCGGCCA1269                          GlyArgPheSerSerPro                                                            375                                                                           TTTCCCTCTTCCTGTGTCCCCAGATCCCCACTACAGCTCCAAGAGGATGGGCCTAGAAAG1329              CCAAGTGCAAAGATGAGGGCAGATTCTTTACCTGTCTGCCCTCATGATTTGCCAGCATGA1389              ATTC1393                                                                      (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 376 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetGluThrLeuSerAsnAlaSerGlyThrPheAlaIleArgLeuLeu                              151015                                                                        LysIleLeuCysGlnAspAsnProSerHisAsnValPheCysSerPro                              202530                                                                        ValSerIleSerSerAlaLeuAlaMetValLeuLeuGlyAlaLysGly                              354045                                                                        AsnThrAlaThrGlnMetAlaGlnAlaLeuSerLeuAsnThrGluGlu                              505560                                                                        AspIleHisArgAlaPheGlnSerLeuLeuThrGluValAsnLysAla                              65707580                                                                      GlyThrGlnTyrLeuLeuArgThrAlaAsnArgLeuPheGlyGluLys                              859095                                                                        ThrCysGlnPheLeuSerThrPheLysGluSerCysLeuGlnPheTyr                              100105110                                                                     HisAlaGluLeuLysGluLeuSerPheIleArgAlaAlaGluGluSer                              115120125                                                                     ArgLysHisIleAsnThrTrpValSerLysLysThrGluGlyLysIle                              130135140                                                                     GluGluLeuLeuProGlySerSerIleAspAlaGluThrArgLeuVal                              145150155160                                                                  LeuValAsnAlaIleTyrPheLysGlyLysTrpAsnGluProPheAsp                              165170175                                                                     GluThrTyrThrArgGluMetProPheLysIleAsnGlnGluGluGln                              180185190                                                                     ArgProValGlnMetMetTyrGlnGluAlaThrPheLysLeuAlaHis                              195200205                                                                     ValGlyGluValArgAlaGlnLeuLeuGluLeuProTyrAlaArgLys                              210215220                                                                     GluLeuSerLeuLeuValLeuLeuProAspAspGlyValGluLeuSer                              225230235240                                                                  ThrValGluLysSerLeuThrPheGluLysLeuThrAlaTrpThrLys                              245250255                                                                     ProAspCysMetLysSerThrGluValGluValLeuLeuProLysPhe                              260265270                                                                     LysLeuGlnGluAspTyrAspMetGluSerValLeuArgHisLeuGly                              275280285                                                                     IleValAspAlaPheGlnGlnGlyLysAlaAspLeuSerAlaMetSer                              290295300                                                                     AlaGluArgAspLeuCysLeuSerLysPheValHisLysSerPheVal                              305310315320                                                                  GluValAsnGluGluGlyThrGluAlaAlaAlaAlaSerSerCysPhe                              325330335                                                                     ValValAlaGluCysCysMetGluSerGlyProArgPheCysAlaAsp                              340345350                                                                     HisProPheLeuPhePheIleArgHisAsnArgAlaAsnSerIleLeu                              355360365                                                                     PheCysGlyArgPheSerSerPro                                                      370375                                                                        (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (primer)                                              (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..21                                                           (D) OTHER INFORMATION: /standard.sub.-- name= "ZC6657"                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GCGGAATTCGAATCACAGGTT21                                                       (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (primer)                                              (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..20                                                           (D) OTHER INFORMATION: /standard.sub.-- name= "ZC6658"                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       ATAGAATTCATCGCATTTCC20                                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (primer)                                              (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..24                                                           (D) OTHER INFORMATION: /standard.sub.-- name= "ZC6770"                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       TCTCTTCTCACCGAAGTGAACAAG24                                                    (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (primer)                                              (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..24                                                           (D) OTHER INFORMATION: /standard.sub.-- name= "ZC6771"                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       TTCTGTCTTTTCAGCTACCCAGGT24                                                    (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (primer)                                              (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..15                                                           (D) OTHER INFORMATION: /standard.sub.-- name= "ZC2682"                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GGTAGCGACCGGCGC15                                                             (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (primer)                                              (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..15                                                           (D) OTHER INFORMATION: /standard.sub.-- name= "ZC2683"                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GACTCCTGGAGCCCG15                                                             (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (primer)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      CTCCATTCTGCTGCACCGGGAATTCCTGACCACAGCAGT39                                     (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (primer)                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      AGATTCCATGCAGCACTCTGCAACTACAAAGCAGCTCGA39                                     (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      AATTAGGGAGACCGGAATTCTGTGCTCTGTCAA33                                           (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      AATTTTGACAGAGCACAGAATTCCGGTCTCCTT33                                           (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      AATTGAGCTCG11                                                                 (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      AATTCGAGCTC11                                                                 __________________________________________________________________________

What is claimed is:
 1. An isolated nucleic acid encoding a mammalianCAP-3 protein wherein said protein is at least 80% identical to theamino acid sequence depicted in Seq ID No. 4, and inhibits serineprotease activity.
 2. The isolated nucleic acid molecule of claim 1,wherein said CAP-3 protein is a human CAP-3 protein.
 3. The isolatednucleic acid of claim 1, wherein said CAP-3 protein comprises the aminoacid sequence depicted in Seq. ID No. 4 or an allelic variant thereof.4. The isolated nucleic acid molecule of claim 1, which is DNA.
 5. Anexpression vector comprising the following operably linked elements:atranscriptional promoter; a DNA segment encoding a mammalian CAP-3protein wherein said protein is at least 80% identical to the amino acidsequence depicted in Seq. ID No. 4, and inhibits serine proteaseactivity; and a transcriptional terminator.
 6. The expression vectoraccording to claim 1, wherein the DNA segment encodes the amino acidsequence depicted in Seq. ID No. 4 or an allelic variant thereof.
 7. Acultured host cell transformed or transfected with an expression vectorwhich comprises the following operably linked elements:a transcriptionalpromoter; a DNA segment encoding a mammalian CAP-3 protein wherein saidprotein is at least 80% identical to the amino acid sequence depicted inSeq. ID No. 4, and inhibits serine protease activity; and atranscriptional terminator.
 8. The host cell of claim 7, which is amammalian cell.
 9. A method for producing a mammalian CAP-3 protein,which comprises:growing eukaryotic cells transformed or transfected witha DNA construct which comprises the following operably linkedelements:(i) a transcriptional promoter (ii) a DNA segment encoding amammalian CAP-3 protein wherein said protein is at least 80% identicalto the amino acid sequence depicted in Seq. ID No. 4, and inhibitsserine protease activity, and (iii) a transcriptional terminator, underconditions whereby said DNA segment is expressed.
 10. The methodaccording to claim 9, further comprising the step of isolating themammalian CAP-3 protein from the cells.
 11. The method of claim 9,wherein the cells are cultured mammalian cells.
 12. The method of claim9, wherein the mammalian CAP-3 protein is isolated by immunoaffinitypurification or by affinity purification using a serine protease whichis a CAP-3 substrate.